1. Field of the Invention
The present invention relates to a method for measuring analyte substance(s) and the apparatus therefor, and in particular to a method and apparatus suited for the quantitative immunoassay of specific substances contained in trace amounts in a multi-component solution such as specimens from biological sources and the apparatus therefor. While the present invention will hereinbelow be illustrated only for measurement of biosubstances in clinical examination, the method and apparatus of the present invention are applicable to a wide variety of fields such as pharmacy, biology, zoology, botany, agriculture and chemistry.
2. Description of the Prior Art
Substances participating in bioactivity of a living body are generally present in trace amounts, and still many of them play very important roles for the living body. The quantitative determination of such bioactive substances therefore is important in fields associated with living organisms, such as medical science and biochemistry. Various methods for that purpose have been proposed and put to practical use, among which a determination method of analytereceptor system utilizing enzymes, radioactive isotopes, chemiluminescent substances or the like as a label has been widely used. The analyte-receptor-type measurement comprises first contacting a solid phase having fixed a first receptor which can specifically bind an analyte which is the substance to be determined, with a sample solution and a labelled second receptor or a labelled analyte (hereinafter these labelled substances are referred to as "the conjugates") simultaneously or successively, thereby effecting an analyte-receptor reaction, washing the reaction mixture, and thereafter determining the amount of the conjugates remaining on the solid phase to measure the amount of the analyte in the sample solution. In this case substances having a large amplification effect such as radioisotopes and enzymes are used as the label. As regards the receptor, for an antigen or a hapten, an antibody which can specifically bind it is used; for an antibody, its antigen is used; for a DNA or RNA, another DNA or RNA complementary thereto is used; and for a ligand, its receptor is used. The heterogeneous EIA, so-called Enzyme Linked Immuno Sorbent Assay (ELISA) is known as a representative example of such determination.
In ELISA, a solid phase obtained by fixing a receptor capable of specifically binding the analyte to be determined, on a test tube, microplate or the like is used for capturing the substance to be determined in the sample solution. An enzyme is used as the label for signal amplification. For example where the analyte to be determined is an antigen, the sandwich-ELISA uses an enzyme-labelled second antibody (a second receptor) bindable to the antigen, and the competitive-ELISA uses an enzyme-labelled antigen as the conjugate. On the other hand, where the analyte to be determined is an antibody, the sandwich-ELISA uses an antigen as the receptor, and another antigen labelled with an enzyme as the second receptor. The competitive system utilizes an antigen as the receptor, selecting an antibody which competes with the antibody to be determined against the antigen, and labels the antibody thus selected with an enzyme. After being washed, the solid phase is contacted with a substrate solution for the above-mentioned enzyme used as the label and, if necessary, a chromophore, whereby the optical property of the system is changed with the proceeding of the decomposition reaction of the substrate, which change is then observed.
Several methods have been used for the purpose of observing the change of the optical property of the substrate solution. Among them, there are methods utilizing optical instruments such as optical absorption instruments fluorophotometer, and chemiluminescence photometer (see for example Ishikawa, Kawai and Miyai, ENZYME IMMUNOASSAYS, 1982, published from Igaku Shoin, Tokyo).
There is also a method which comprises comparing a substrate solution with a control solution and observing visually the difference in the color to judge the presence of a trace amount of an analyte substance (see for example Japanese Patent Application Laid-Open No. 128369/1985).
The above-mentioned optical measuring systems utilizing optical instruments, however, require expensive and large and complex equipment because they generally need a stable light source, a photometer having high sensitivity, a photomultiplier circuit with high precision, and the like. Further, specialized technicians must attend to the measurement since special technique is required for the measurement.
On the other hand, the direct visual observation method is a qualitative one. It is liable to personal variation in judgement of color change and to the observer's subject. Further, judgement itself is sometimes difficult for measurement of an extreme trace substance where the color change is very small.